The long term goals for this grant have been to elucidate the structure and function of the regulatory subunits of PKA. Initially, we focused on protein chemistry and sequencing to identify functional sites. Structures of Rla in 1995 and of Rllp in 2001 defined a conservedfold and also revealed remarkable isoform diversity. During this past granting period we focused on developing techniques such as H/D exchange and small angle Xray scattering to define the dynamic properties of these proteins in solution. Most recently, we solved the first structure of a complex of an Rla subunit and bound to C. C serves as a stable scaffoldfor docking Rla while Rla undergoes remarkable conformational changes. Our goals for the next granting period are to probe the structure, function, and dynamics of the regulatory subunits focusing primarily on Rla and Rllp. Our specific aims are to achieve a molecular understanding of cAMP binding and kinase inhibition. With a structure of an Rla:C complex and a general model for cAMP binding domains in hand, we can now probe these structures more comprehensively than ever before. The cyclic nucleotide binding domain is a major signaling motif that has been conservedthroughout evolution. Thus the rules that we define for cAMP binding will have general applicability for this universal sensing module. On the other hand, the specific mechanisms that we elucidate for inhibition of kinase activity will also have broad relevance for the protein kinase family. We have four Specific Aims. Our first aim is to define and comprehensively map the molecular features of the cAMP binding domains in Rla and Rll|3 using a combination of mutagenesis, biochemistry, and biophysics as well as H/DMS. Our second aim is to characterize the dynamic features of Rla and Rllp that are associated with cAMP binding andholoenzyme dissociation using fluorescence anisotropy, stopped flow fluorescence, and fluorescence resonance energy transfer (FRET). The third aim is to solve structures that reflect the different conformational states of these proteins using small angle Xray scattering and neutron scattering to obtain low resolution information in solution and Xray crystallography for high resolution data. The fourth aim is to develop NMR techniques to provide an atomic level understanding of Rla dynamics using Rla(119-244) and Rla(91-244) as prototypes. General significance. Understanding howcells convert an extracellular signal into a biological response is central to all of biology. With PKA, a ubiquitous cAMP-dependent protein kinase that regulates processes as diverse as memory and development, wehave the potential to unravel a fundamental signaling response in great molecular detail. Thework willnot only enhance our basic understanding of these molecular switches but will also open the door to the design of new therapeutics capable of interfering with cell signaling.